This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Previous studies of oxidation of leucine-containing peptides revealed the formation of a peptidyl hydroperoxide on the leucine side chain. Peptidyl hydroperoxides have never before been observed in hydroxyl radical footprinting experiments. It was previously thought that they were too unstable to survive the protein preparation and mass spectrometry analysis;however, their discovery in the leucine-containing peptide by mass spectrometry indicates that they are sufficiently stable to persist in solution. If they do exist in solution, they may be capable of performing direct two-electron oxidation of sulfur-containing side chains, causing false positives in hydroxyl radical footprinting experiments. Additionally, standard methods used to remove hydrogen peroxide, including catalase treatment and liquid chromatography would be ineffective at removing peptidyl hydroperoxides. We designed and carried out a series of experiments to determine if peptidyl hydroperoxides are common products of oxidation of various amino acids, to determine if these peptidyl hydroperoxides are capable of two-electron oxidation of methionine residues, and to determine if catalase treatment or scavenging with methionine amide is effective at eliminating the peptidyl hydroperoxides.